Membrane Protein Crystallization and Structure Determination in Lipidic Cubic Phase or Sponge Mesophase: Membrane proteins constitute 30% of the genome, and are targets for over 40% of all drugs in use today, however only in the past year have structural interactions of the first eukaryotic membrane protein with a drug emerged. Atomic level structures define mechanisms of action, and greatly enhance drug discovery against this untapped therapeutic landscape. The means of structure determination is X-ray crystallography requiring high quality crystalline membrane proteins. Most of the membrane proteins crystallized to date have been crystallized using detergents to solubilize the membrane protein out of its lipid environment. An alternative approach is to crystallize using more natural lipid environments using 'mesophases'of lipids. However until recently the Lipidic Cubic Phase (LCP) methods pertained only to small colored proteins from archaebacteria. A more hydrated and open mesophase, of reduced interfacial curvature supports facile crystallization of larger and bulkier proteins. This method has yielded structure- grade crystals of the light-harvesting II complex to 2.45 [unreadable] resolution with dramatically higher packing density than by detergent methods. To facilitate rapid, economic screening using this method, Dr. Martin Caffrey adapted a robotic system to high throughput application of this method that is otherwise intractable for the majority of membrane proteins. The robot uses a standard laboratory fluid handling robot modified by addition of an accurate dispenser of viscous mesophases in 30 nanoliter volumes augmented by 20 nL protein and 800 nL precipitant solution for each trial. 96 wells can be set up using the robot in 9 minutes. Evaluation of crystallization progress is by an imaging system enabling a 96-well plate to be imaged in 15 minutes. Through primary grants we each produce pure homogeneous and stable membrane proteins calibrated for crystallization. To date the Stroud group has determined structures of 15 membrane proteins maintained in detergent. This robot will allow a different type of crystallization of membrane proteins in membrane lipids. The method has been used with landmark success by the Stevens group at Scripps, using Caffrey's robot, that led to structures of 2-adrenergic receptors (Cherezov, V., et al. Science 318: 1258-65, 2007). Caffrey provided designs to Anachem and is a collaborator with us on our joint renewal of Roadmap proposal GM 73210. To date there are only two of these instruments in use;this would be a third, built under the guidance of Martin Caffrey, by Anachem, Ltd and distributed by Zinsser North America, Inc. This robot enables us to tap into the most promising new method of crystallization of membrane proteins in a high throughput format. As such it opens the horizon to crystallize many more membrane proteins under defined exact conditions that simulate the natural environment, the lipid bilayer. HEALTH RELEVANCE: The Lipidic Cubic Phase robotic instrument facilitates structure determination of Membrane Proteins in their lipid environments. Membrane proteins constitute ~30% of all eukaryotic proteins and are targets for over 40% of all drugs in use today, however only in the past year have structural interactions of the first eukaryotic membrane protein with a drug emerged. This proposal seeks to advance structural determination of membrane proteins in general, and targets human or pathogenic membrane proteins of importance to human health.